This invention relates to a method of introducing substances into living cells or tissues without killing those cells or tissues.
Biologists often need to introduce into living cells or tissues a wide range of substances which are normally excluded from the cell by the cell walls and outer membranes. These substances include biological stains, proteins, nucleic acids, organelles, chromosomes, and nuclei. One application of central importance is the introduction of genetic materials into cells for the purpose of genetic engineering.
Sanford et al., U.S. Pat. No. 4,945,050, (incorporated herein by reference), discloses a method of transporting substances into living cells and tissues, and an apparatus therefor. Generally, the method disclosed in Sanford et al. involves propelling inert or biologically active particles at cells at a sufficient speed that the particles penetrate the surface of the cells and become incorporated into the interior of the cells. These particles thus provide a vector for introducing substances into living cells. Sanford et al. discloses various apparatus for accelerating the particles to a predetermined speed and propel the particles toward a target.
While the Sanford et al. method is effective, it has a number of shortcomings. The literature reports that in micro projectile bombardment there is poor control over size, aggregation, coating, quantity, dispersal and velicity of particles. A principal shortcoming of Sanford, et al. is that the method is not particularly efficient. It is believed that at least some of the weakly adherent coating of genetic material is lost in the process of accelerating the particles to the speed required to penetrate the cell walls. It is also believed that at least some of the coating of genetic material is "wiped" off the particles as they penetrate the cell walls. Another shortcoming of Sanford, et al. is that it is not satisfactory for introducing RNA, which is unstable, into cells. It is difficult to coat particles with RNA without altering or destroying the RNA. The inventor also believes that biological material, such as DNA is better able to withstand the acceleration and other forces encountered in the introduction into living cells, when in a liquid droplet, rather than on a solid particle. Still another shortcoming of Sanford, et al. is the physical disruption of the cell caused by the particle, and the heat generated by the impact of the particle on the cell. Finally, the ability to adjust particle energy is limited to Sanford, et al., making adjustments to different cell type difficult.